Proc. Natl. Acad. Sci. USAVol. 93, pp. 3275-3279, April 1996Cell Biology

DUB-1, a deubiquitinating enzyme with growth-suppressing activity

(cytokines/interleukin 3/ubiquitin/cell cycle)

YUAN ZHU*, MARTIN CARROLL*, FEROZ R. PAPAt, MARK HOCHSTRASSERt, AND ALAN D. D'ANDREA***Divisions of Pediatric Oncology and Cellular and Molecular Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; andtDepartment of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637

Communicated by David M. Livingston, Dana-Farber Cancer Institute, Boston, MA, November 29, 1995 (received for review October 3, 1995)

ABSTRACT Cytokines regulate cell growth by inducingthe expression of specific target genes. Using the differentialdisplay method, we have cloned a cytokine-inducible imme-diate early gene, DUB-I (for deubiquitinating enzyme).DUB-I is related to members of the UBP superfamily ofdeubiquitinating enzymes, which includes the oncoproteinTre-2. A glutathione S-transferase-DUB-I fusion proteincleaved ubiquitin from a ubiquitin-j3-galactosidase protein.When a conserved cysteine residue of DUB-I, required forubiquitin-specific thiol protease activity, was mutated toserine (C60S), deubiquitinating activity was abolished. Con-tinuous expression of DUB-I from a steroid-inducible pro-moter induced growth arrest in the G1 phase of the cell cycle.Cells arrested by DUB-1 expression remained viable andresumed proliferation upon steroid withdrawal. Our resultssuggest that DUB-1 regulates cellular growth by modulatingeither the ubiquitin-dependent proteolysis or the ubiquitina-tion state of an unknown growth regulatory factor(s).

Interleukin 3 (IL-3) is a glycoprotein hormone that regulatesgrowth of hematopoietic progenitor cells (1). IL-3, like othercytokines, acts during the G1 phase of the cell cycle to drivecells into S phase. IL-3 exerts its biologic function through aspecific receptor (IL-3R) that is expressed on its target cells (2,3). The IL-3R activates multiple signal transduction pathways,including the Ras-Raf-mitogen activating protein kinase path-way and the JAK-STAT pathway, resulting in the induction ofimmediate early genes. How these immediate early genescouple IL-3R activation to the biochemical machinery of cellgrowth and cell cycle progression is poorly understood.

Cell growth and cell cycle progression are controlled, at leastin part, by ubiquitin-mediated proteolysis (4, 5). Ubiquitin-mediated proteolysis requires ATP and results in covalentconjugation of target proteins with multiple ubiquitin mole-cules (6-9). Multiubiquitinated proteins are rapidly degradedby the 26S proteasome, a multicatalytic protease complex (10,11). Recent evidence shows that intracellular levels of cyclinsand cyclin dependent kinase inhibitors (12, 13), as well as othergrowth regulatory proteins, such as p53 (14, 15), c-Jun (16),and IKBa (17), are regulated by ubiquitin-mediated proteol-ysis. It is also possible that ubiquitination alters a protein'sfunction without affecting its metabolic stability (18).

Little is known about the regulatory enzymes that determinewhich cellular proteins are specifically destroyed by ubiquitin-mediated proteolysis. Most evidence suggests that substratespecificity is determined by ubiquitin-conjugating enzymes(19, 20). Recently, a large superfamily of genes encodingdeubiquitinating enzymes was identified (21). Deubiquitinat-ing enzymes remove ubiquitin from intracellular protein con-jugates by cleaving the amide linkage between the C terminusof ubiquitin and either a-amino or E-amino groups of the

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substrate. These enzymes are ubiquitin specific but sharecertain properties with other thiol proteases. Genes for at least15 deubiquitinating enzymes were identified from the yeastgenome, making them the largest known gene family in theubiquitin system. Several proteins implicated in growth anddevelopment, including the mammalian proteins Tre-2 andUnp and the Drosophila fat facets protein, were either shownto be deubiquitinating enzymes or to have sequence similarityto such enzymes (21).

In the current study, we used the strategy of differentialdisplay (22, 23) to clone an immediate early cDNA (DUB-1)that is specifically induced by IL-3. The DUB-1 cDNA encodesa 526-aa protein that has deubiquitinating activity. Interest-ingly, misregulated expression of DUB-1 induces cell cyclearrest in the G, phase of the cell cycle. Our results support thehypothesis that protein ubiquitination is important in growth-factor-mediated cellular proliferation. They also implicatedeubiquitinating enzymes as regulatory enzymes that coupleextracellular signaling to cell growth.

MATERIALS AND METHODSCells and Cell Culture. Ba/F3 is an IL-3-dependent murine

pro-B cell line (24). Ba/F3 cells were maintained in RPMI1640 medium supplemented with 10% fetal calf serum (FCS)and 10% conditioned medium from WEHI-3B cells as a sourceof IL-3 (25).

Differential Display and Cloning of DUB-1 cDNA. Totalcellular RNA was isolated from starved or IL-3-stimulatedBa/F3 cells by the guanidinium isothiocyanate procedure (26)and subjected to the differential display analysis (22) (GeneHunter, Boston). A partial cDNA fragment that was specifi-cally induced by IL-3 was isolated using a 5' primer (5'-TCTGTGCTGG-3') and a 3' primer (5'-TTTTTTTTTTT-TGT-3') and subcloned into pCRII (Invitrogen). This partialcDNA (298 bp) was shown by dideoxy DNA sequencing tocontain the 5' and 3' primers. A cDNA library, from Ba/F3cells growing in IL-3, was constructed in the phage vectorAZAP (Stratagene). Poly(A)+ mRNA used for library con-struction was prepared by the Fast Track mRNA Isolation Kit(Invitrogen). The partial cDNA isolated by differential displaywas labeled with [32P]dCTP by random prime labeling (27) andused to screen 1 x 106 plaque-forming units from the library.Three independent positive clones of different lengths thathybridized with the probe were isolated, and the correspondingplasmids were isolated from the phage clones. The longestcDNA clone was sequenced on both strands by the dideoxyDNA sequencing method (United States Biochemical).

Abbreviations: IL-3, interleukin 3; IL-3R, IL-3 receptor; GST, gluta-thione S-transferase; ORF, open reading frame.ITo whom reprint requests should be addressed at: Division ofPediatric Oncology, Dana-Farber Cancer Institute, 44 Binney Street,Boston, MA 02115.

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Northern Blot Analysis. RNA samples (10-30 jig) wereelectrophoresed on denaturing formaldehyde gels and blottedonto Duralon-UV membranes (Stratagene). The cDNA in-serts, purified from agarose gels (Qiagen, Chatsworth, CA),were radiolabeled (27) and hybridized for 1 hr to the filters ina 68°C oven. Hybridized filters were finally washed at roomtemperature in 0.1 x SSC (1 x SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7) and 0.1% SDS.

Deubiquitination Assay. The deubiquitination assay of ubiq-uitin-p3-galactosidase fusion proteins has been previously de-scribed (21). A 1578-bp fragment from the wild-type DUB-1cDNA (corresponding to aa 1 to 526) and a cDNA containinga missense mutation (C60S) were generated by polymerasechain reaction (PCR) and inserted, in frame, into pGEX-2TK(Pharmacia) downstream of the glutathione S-transferase(GST) coding element. Ub-Met-f3gal was expressed from apACYC184-based plasmid. Plasmid-bearing Escherichia coliMC1061 cells were lysed and analyzed by immunoblotting withanti-f3gal antibodies (Cappel) and the enhanced chemilumi-nescence system (Amersham).

Generation of Anti-DUB-I Antiserum and Analysis of theDUB-I Polypeptide. A DUB-1 antiserum was raised by inject-ing a full-length GST-DUB-1 fusion protein into a NewZealand White rabbit and was affinity purified with a GST-DUB-1 affinity matrix, as previously described (28). In vitrotranslation of the full length DUB-1 polypeptide was per-formed by standard procedures (Promega). Immunoblottingwas performed as previously described (29) using the affinity-purified anti-DUB-1 antiserum and enhanced chemilumines-cence technology.

Heterologous Expression ofDUB-I in Ba/F3 Cells and CellGrowth Analysis. The open reading frame (ORF) of DUB-1[or DUB-1(C60S)] was generated by PCR using the followingprimers: 5'-GCGAATTCTTTGAAGAGGTCTTTGGAGA-3' (-19 to 1) and 5'-ATCTCGAGGTGTCCACAGGAGCCT-GTGT-3' (1802 to 1781). The fragments (1637 bp) weresubcloned into the Sma I/Xho I cloning sites of pMSG(Pharmacia), which contains a mouse mammary tumor virus-long terminal repeat inducible promoter and a gpt selectionmarker. Parental Ba/F3 cells were electroporated with vectoralone or with pMSG-DUB-1 as previously described (25).After 3 days in IL-3 medium, the cells were selected in IL-3medium containing 250 /ag/ml xanthine, 15 j/g/ml hypoxan-thine, 10 /tg/ml thymidine, 2 /ug/ml aminopterin, and 25jig/ml mycophenolic acid. Gpt-resistant subclones were iso-lated by limiting dilution. DUB-1 expression was induced byadding 0.1 ,uM dexamethasone (diluted from 10 mM stock inethanol). Cell proliferation and cell viability were measured bytrypan blue exclusion (25).

Analysis of Cell Cycle. Cell cycle analysis was performed byfluorescence-activated cell sorter, as previously described (30).The percentage of cells in each phase of the cell cycle wasdetermined by analyzing data with the computer programCELLFIT (Becton Dickinson).

RESULTSDUB-I Is a Hematopoietic-Specific Immediate Early Gene

Encoding a Deubiquitinating Enzyme. Ba/F3 is a murinelymphocyte cell line that depends on IL-3 for growth andviability (24,30,31). By comparing mRNA from IL-3-deprivedand IL-3-stimulated Ba/F3 cells (22, 23), we initially isolatedan IL-3 inducible, immediate early cDNA fragment (DUB-I).The full-length 2674-bp DUB-1 cDNA was subsequently iso-lated and found to contain a 1581-bp ORF (Fig. 1A). There aretwo stop codons within the 183 bp of 5' untranslated region.In addition, we isolated a murine genomic clone that containsa TATA box at position -321 and an IL-3 inducible enhancer(Y.Z., unpublished data).

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GAATTCGGCACGAGGAAAAACTTCCTTCTGCTCCCTTAGAAGACTCCAGCTAGTTATTTGAAGAGGTCTTTGTAGACACGGTGGTTGCTCTTTCCTCCCAAGAAGAGATTCTCTAGAAGGGAAAAACTTCCTTCTGCTCCCTTAGAAGACTACAGCAAGTTCTTTGAAGAGGTCTTTGGAGACATGGTGGTTGCTCTTTCCTTCCCAGAAGCAGATCCAGCCCTATCATCTCCTGATGCC

M V V A L S F P E A D P A L S S P D ACCAGAGCTGCATCAGGATGAAGCTCAGGTGGTGGAGGAGCTAACTGTCAATGGAAAGCACP E L H Q D E A Q V V E E L T V N G K HAGTCTGAGTTGGGAGAGTCCCCAAGGACCAGGATGCOGGCTCCAGAACACAGGCAACAGCS L S W E S P Q G P G C G L Q N T G N STGCTACCTGAATGCAGCCCTGCAGTGCTTGACACACACACCACCTCTAGCTGACTACATGC Y L N A A L Q C L T H T P P L A D Y MCTGTCCCAGGAGCACAGTCAAACCTGTTGTTCCCCAGAAGGCTGTAAGTTGTGTGCTATGL S Q E H S Q T C C S P E G C K L C A MGAAGCCCTTGTGACCCAGAGTCTCCTGCACTCTCACTCGGGGGATGTCATGAAGCCCTCCE A L V T Q S L L H S H S G D V M K P SCATATTTTGACCTCTGCCTTCCACAAGCACCAGCAGGAAGATGCCCACGAGTTTCTCATGH I L T S A F H K H Q Q E D A H E F L MTTCACCTTGGAAACAATGCATGAATCCTGCCTTCAAGTGCACAGACAATCAAAACCCACCF T L E T M H E S C L Q V H R Q S K P TTCTGAGGACAGCTCACCCATTCATGACATATTTGGAGGCTGGTGGAGGTCTCAGATCAAGS E D S S P I H D I F G G WW R S Q I KTGTCTCCTTTGCCAGGGTACCTCAGATACCTATGATCGCTTCCTGGACATCCCCCTGGATC L L C Q G T S D T Y D R F L D I P L DATCAGCTCAGCTCAGAGTGTAAAGCAAGCCTTGTGGGATACAGAGAAGTCAGAAGAGCTAI S S A Q S V K Q A L W D T E K S E E LTGTGGAGATAATGCCTACTACTGTGGTAAGTGTAGACAGAAGATGCCAGCTTCTAAGACCC G D N A Y Y C G K C R Q K M P A S K TCTGCATGTTCATATTGCTCCAAAGGTACTCATGGTAGTGTTAAATCGCTTCTCAGCCTTCL H V H I A P K V L M V V L N R F S A FACGGGTAACAAGTTAGACAGAAAAGTAAGTTACCCGGAGTTCCTTGACCTGAAGCCATACT G N K L D R K V S Y P E F L D L K P YCTGTCTGAGCCTACTGGAGGACCTTTGCCTTATGCCCTCTATGCCGTCCTGGTCCATGATL S E P T G G P L P Y A L Y A V L V H DGGTGCGACTTCTCACAGTGGACATTACTTCTGTTGTGTCAAAGCTGGTCATGGGAAGTGGG A T S H S G H Y F C C V K A G H G K WTACAAGATGGATGATACTAAAGTCACCAGGGTGTGATGTGACTTCTGTCCTGAATGAGAATY K M D D T K V T R C D V T S V L N E NGCCTATGTGCTCTTCTATGTGCAGCAGGCCAACCTCAAACAGGTCAGTATTGACATGCCAA Y V L F Y V Q Q A N L K Q V S I D M PGAGGGAAGAATAAATGAGGTTCTTGACCCTGAATACCAGCTGAAGAAATCACGGAGAAAAE G R I N E V L D P E Y Q L K K S R R KAAGCATAAGAAGAAAAGCCCTTTCACAGAAGATTTAGGAGAGCCCTGCGAAAACAGGGATK H K K K S P F T E D L G E P C E N R DAAGAGAGCAATTAAAGAAACCTCCTTAGGAAAGGGGAAAGTGCTTCAGGAAGTGAACCACK R A I K E T S L G K G K V L Q E V N HAAGAAAGCTGGGCAGAAACACGGGAATACCAAACTCATGCCTCAGAAACAGAACCACCAGK K A G Q K H G N T K L M P Q K Q N H QAAAGCTGGGCAGAACCTCAGGAATACTGAAGTTGAACTTGATCTGCCTGCTGATGCAATTK A G Q N L R N T E V E L D L P A D A IGTGATTCACCAGCCCAGATCCACTGCAAACTGGGGCAGGGATTCTCCAGACAAGGAGAATV I H Q P R S T A N W G R D S P D K E NCAACCCTTGCACAATGCTGACAGGCTCCTCACCTCTCAGGGCCCTGTGAACACTTGGCAGQ P L H N A D R L L T S Q G P V N T W QCTCTGTAGACAGGAAGGGAGACGAAGATCGAAGAAGGGGCAGAACAAGAACAAGCAAGGGL C R Q E G R R R S K K G Q N K N K Q GCAGAGGCTTCTGCTTGTTTGCTAGTGATCACCCACCCACTCACACAGGCTCCTGTGGACAQ R L L L V C *CACTGTTGACCCAAGGTGCCTGGAACAAGAGGTTTGGATCTCTGTTTCAGGCAGGGACAATGCCTCACCCTTCACGTGGGGTCCACCTATCCTCTGGGCCCTTGCCTGTTTTTGCTGACTGACTCTCTGATTGTTTGAATGTGGAAAAAAAGTGCCCAGGATGTTGGTACAGGTTAAAGACAAGAAGCTGGACACCCGGAGGAGGTCTGAATAGCCTCTCCTGCAACTCATGGAATCTGAGCAGCATAGAGACTAAATCACCACACTGGAGCTTTCTTTTCTTTTCTTTTCTTTTCTTTTCTTTTCTTTTCTTTTCTTTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTTTCCTTTCCTTTCCTTTCCTTTTTTTTAAATTTTTTGTTATTAGATATTTTCTTTATTTACATTTCAAATGCTATCCCAAAAGTTCCCTATACCCTCCCCCAACTCTGCCACCCTACCCACCCACTCCCACTTCTTGGCTCTGGCATTTCCCTGTACTGGGGCATATAAAGTTTGCAATACCAAAGGGCCTCTCTTCCCAATGATGGCCAACTAGGTCACCTTCTGCTACATATGCAGCTAGAGACCCTAAGAAAACACACTGGAACTCTTGAGGTTTGGAGTTTTCGCTCAGGCAAACAAGTTGCTTTTCAACTGCCCTTTCTAACCTCTTACCCAGAAAATGTGTAGTTCACCCTGTAGAGATAGATGCTCTTATTCTTAGTGTGTGATCAACAGTTCTTTGGTCA,AA TTCTGTTACTTCACAAAAAAAAAAAAAA 2674

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B Cys domaintre-2 216 G[TGL LtG T C F M N S A LTQ C L 233Unp 135 G L S N L G N T C F M N S S I Q C V 152Doa4 563 G L E N L G N i C Y M N C I I Q C 1 580Dub-1 52 G L QN iGN S C Y L N A A L 69

His domaintre-2 995 Y N L I S C H S G I L S - G G H Y I Tf A K N P - N CUnp 696 Y D LllA V S N H Y G A M G - V G H Y T A Y A K N R L N GDoa4 864 Y E L VYLV A C H F G T L Y - G G H Y T A YV K K G L K K

Dub-1 290 Y A L Y A V L V H D G A T S H S G H Y F C C V K A G - H G

K W YC Y N S S CE E L - H P D E I D TD S TYIL F Y 1049KWJYY F D D S S V S L A - S E D Q I V TK A A Y V L F Y 751WI.Y F D D T K Y K P V K N K A D A I NS N A Y V L F Y 920

KWY M D D T K VT R C - D V T S V L NEN A Y V L F Y 345

Myc homologyMyc 313rnS T R Kn Y PrAAKl R A K|TD SFRrTT1 K QlT S N N R 340Dub-1 394WP]C E N RUK RAI E TSIEJGKG KLV LjQ EUJ N H K K 421FIG. 1. Sequence and homologies of the DUB-1 cDNA. (A) Nucle-

otide and predicted amino acid sequence of DUB-1. Underlined se-quences are copies of a conserved motif shown by Shaw and Kamen (32)to confer message instability and which are found in the 3' untranslatedregions of many mitogen-induced, immediate early mRNAs. A consensuspolyadenylylation signal is double underlined. The sequence of themurine DUB-1 cDNA has been assigned GenBank no. 24133 U41636. (B)Sequence homologies between yeast Doa4 (21), human Tre-2 (33),murine Unp (34), and murine DUB-1. Alignment ofDUB-1 with humanc-myc is also shown. The homologous domain of c-myc contains thenuclear localization sequence PAAKRAKLD (35) but not the c-mycDNA binding domain.

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The DUB-1 ORF is predicted to encode a polypeptide of 526aa (59 kDa). Comparison of the DUB-1 protein sequence withentries in GenBank data base (3/96) detected significantsimilarity with several deubiquitinating enzymes, includingTre-2 (33, 36), Unp (34), and Doa4 (21). The sequencesimilarity was largely restricted to the conserved Cys and Hisboxes previously identified for this enzyme superfamily (Fig.1B) (21). These elements probably help form the enzymeactive site (21). The likely active site nucleophile is a cysteineresidue in the Cys box that is found in all known familymembers (21) and is also present in DUB-1 (Cys60). The 3'untranslated region of the DUB-1 cDNA contained two AT-TTA sequences, located in A + T rich domains. The AUUUAsequence, found in the 3' untranslated regions of many im-mediate early mRNAs, may play a role in DUB-1 mRNAturnover (32). The DUB-1 mRNA was detected in multiplehematopoietic cell lines, but not in nonhematopoietic cell linesor tissues from adult mice (data not shown).DUB-I Encodes a Functional Deubiquitinating Enzyme. In

order to determine whether DUB-1 has deubiquitinating ac-tivity, we expressed DUB-1 as a GST fusion protein. TheDUB-1 ORF was subcloned into the bacteria expression vector,pGEX. pGEX-DUB-1 was co-transformed into E. coli(MC1061) with a plasmid expressing the protein Ub-Met-/3gal, in which ubiquitin is fused to the N terminus of f3-galac-tosidase. As shown by immunoblot analysis (Fig. 2), twoindependent cDNA clones encoding GST-DUB-1 fusion pro-tein resulted in cleavage of Ub-Met-83 gal (lanes 3, 4, and 7)comparable to that observed with Ubpl, a known yeastdeubiquitinating enzyme (21) (lane 1). As controls, cells withthe pGEX vector (lane 5) or pBluescript vector with a non-transcribed DUB-1 insert (lane 2) failed to cleave Ub-Met-,3gal. A mutant DUB-1 polypeptide, containing a C60S muta-tion, was unable to cleave the Ub-Met-f3 gal substrate (lane 6).Expression of GST-DUB-1 in bacterial cells containing theUb-Leu-j3 gal substrate showed greatly reduced levels of 3 galactivity (data not shown). The Leu-,3 gal product, unlikeMet-/3 gal or the respective Ub-13 gal fusions, is short lived inE. coli (37). This result strongly suggests that DUB-1 cleavesUb-Leu-P3 gal specifically at the C terminus of the ubiquitinmoiety. Taken together, these results demonstrate that DUB-1has deubiquitinating activity and that Cys 60 is critical for itsthiol protease activity.DUB-I mRNA Levels Are Induced by IL-3 in Early G1 Phase,

Followed by a Rapid Decline. Ba/F3 cells arrest in early G1phase when deprived of IL-3 for 12 hr and can be induced toreenter the cell cycle synchronously by readdition of growthfactor (30). The 3.1-kb DUB-1 mRNA appeared 30 to 60 minafter addition of IL-3 (Fig. 3) but rapidly decreased in abun-dance before the completion of G1 phase. DUB-1 mRNA levelswere superinduced with IL-3 plus cycloheximide (data notshown), defining DUB-1 as an immediate early gene. Inductionof DUB-1 mRNA was similar to that of c-myc, although c-mycmRNA levels remained elevated throughout G1 phase. CyclinD2 mRNA accumulated later in G, phase as previouslydescribed (38).

Ub- gal _

Pgai

1 2 3 4 5 6 7

FIG. 2. DUB-1 encodes a functional deubiquitinating enzyme.Deubiquitination of ubiquitin-,3-galactosidase (Ub-Met-j3gal) fusionproteins expressed in bacteria. Shown is a Western blot using anti-,3gal antiserum. Co-expressed plasmids were pGEX-Ubpl (lane 1)(21), pBluescript/DUB-1 (DUB-1 is not expressed) (lane 2), pGEX-DUB-I.1 (lanes 3 and 7), pGEX-DUB-1.2 (lane 4), pGEX(vector)(lane 5), and pGEX-DUB-l(C68S) (lane 6).

Time after stimulation

(hr) 0 .17 0.5 1.0 2 4 6 8 10 12 14 16 18 24

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myc ---- ** OPWFWSF 7'', wxCyclin D2

GAPDH

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G1 S G2/GO/M GI

TIME (hrs) 0 .17 0.5 1.0 2 4 6 8 10 12 14 16 18 24

% GO/GI 94 9394 9394 93 93 89 68 42 39 27 53% S 4 5 4 44 5 2 6 27 56 60 60 39

% G2/M 2 22 2 5 5 5 2 1 14 8

FIG. 3. DUB-1 mRNA levels are induced by IL-3 in early GI phase,followed by a rapid decline. Ba/F3-EPO-R cells were arrested in earlyG, phase by growth factor starvation for 12 hr and were restimulatedwith IL-3 to enter the cell cycle synchronously. Total RNA (10 jug perlane) extracted from cells at the indicated time (in hours) wassubjected to Northern blot analysis with the indicated cDNA probes.The different cell cycle phases were determined by flow-cytometricanalysis of cellular DNA content.

Continuous Expression of DUB-I Arrests Cellular Growth.Our initial attempts to obtain stable cell lines that consti-tutively express DUB-1 were unsuccessful. Because DUB-1expression is normally turned off after only a brief period ofsynthesis (Fig. 3), we reasoned that continuous expression ofDUB-1 mRNA might somehow interfere with cell growthand/or viability. We therefore expressed DUB-1 in Ba/F3cells using an inducible promoter (Fig. 4). Twelve gpt-resistant Ba/F3 subclones were generated after transfectionwith either pMSG/DUB-1 or mutant pMSG/DUB-l(C60S),which encodes the inactive enzyme. Dexamethasone (0.1,uM) induced DUB-1 mRNA in all transfected cells, but notin parental or mock-transfected cells (data not shown).Dexamethasone induced expression of the DUB-1 protein

(Fig. 4A, lane 2) or DUB-1(C60S) protein (lane 4) in trans-fected Ba/F3 cells to levels comparable with those observedduring IL-3-induced expression from the endogenous DUB-1gene (data not shown). These proteins had the same electro-phoretic mobility (59 kDa) as full-length DUB-1 polypeptidesynthesized by in vitro translation (lane 5). After dexametha-sone induction, cells expressing DUB-1 failed to proliferate inIL-3 medium (Fig. 4B). In contrast, dexamethasone-inducedcells expressing DUB-1 (C60S) proliferated normally in IL-3.Importantly, while dexamethasone induction of wild-typeDUB-1 inhibited cellular proliferation, as measured by totalcell number, it had little effect on cellular viability (Table 1).The Ba/F3 subclones that were induced with dexamethasone toexpress either wild-type DUB-1 orDUB-1(C60S) remained viablein IL-3. Cells underwent apoptosis only after removal of IL-3.To test the possibility of nonspecific toxicity caused by pro-

longed expression of active DUB-1 enzyme, we stopped DUB-1synthesis in cells transfected with the wild-type DUB-1 constructby removal of dexamethasone at day 7. Cells resumed normalproliferation within 48 hr following dexamethasone with-drawal (Fig. 4C). To provide further evidence against non-specific toxicity, we induced DUB-1 expression in murine3T3 fibroblasts (data not shown). Normal cell proliferationwas observed for these cells, indicating that growth suppres-sion by DUB-1 is cell-type specific.

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Table 1. Percent viable Ba/F3 cellsIL-3 DEX Day 0 Day 2 Day 3 Day 4

DUB-1 (WT) + - 96 92 88 92+ + 96 89 91 91- - 96 0 0 0- + 96 0 0 0

DUB-1 (C60S) + - 95 90 85 87+ + 95 90 80 82- + 95 0 0 0- - 95 0 0 0

WT, wild type; DEX, dexamethasone.

Normally, DUB-1 mRNA levels rise soon after IL-3 additionduring the early G1 phase of the cell cycle, followed by a rapiddecline. When DUB-1 mRNA levels are maintained by con-tinuous synthesis from a dexamethasone-inducible promoter,Ba/F3 cells arrest in the G1 phase of the cell cycle. These dataindicate that DUB-1 expression is tightly regulated and thatDUB-1 may play a role in cytokine-induced cell proliferation.

Deubiquitinating enzymes studied in yeast have multiplefunctions (21). Some deubiquitinating enzymes, such as Ubp2,can apparently remove ubiquitin from ubiquitin-conjugatedsubstrates prior to proteasome-substrate binding, therebyslowing the turnover of such proteins (39). Other deubiquiti-nating enzymes, such as Doa4, may remove ubiquitin fromproteasome-bound degradation products, allowing recyclingof ubiquitin and proteasomes and thereby promoting furtherprotein degradation (21). Ubiquitin must also be cleaved fromprecursor forms by deubiquitinating enzymes. Finally, dynamicubiquitination events may serve as reversible regulatoryswitches (40, 41).

Failure to turn off expression of DUB-1 presumably, as inour experiments, may cause G, arrest by preventing thedegradation of growth-inhibitory proteins, such as cyclin de-pendent kinase inhibitors, or by promoting the degradation ofgrowth-permissive proteins, such as G, cyclins. Alternatively,

6

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FIG. 4. Continuous expression of DUB-1 results in growth sup-pression. (A) Immunoblot analysis of steroid-induced DUB-1polypeptide. Lysates (100 /ig of total protein) from the indicatedcells were electrophoresed in 10% SDS polyacrylamide gels andblotted with affinity-purified anti-DUB-I antibody (1:1000). (B)Ba/F3-DUB-I (open symbols) or Ba/F3-DUB-I(C60S) (filled sym-bols) were cultured in IL-3 medium with (A, A) or without (o, *)dexamethasone (0.1 /iM). Cell number was calculated by the trypanblue exclusion technique. (C) Ba/F3 cells, transfected with wild-typeDUB-I, were grown in IL-3 with (A) or without (o) dexamethasonefor 6 days. Dexamethasone-treated cells were washed, replated inIL-3 medium (without dexamethasone) on day 7 (arrow), andcultured for an additional 6 days (A).

Cell cycle analysis demonstrated that the majority ofBa/F3 cells were arrested in the G, phase of the cell cyclefollowing dexamethasone induction of DUB-1 (Fig. 5). Thisconcentration of dexamethasone (0.1 /pM) slightly reducedIL-3 dependent proliferation of parental Ba/F3 cells orDUB-1(C60S)-expressing cells (to 80% of maximum), but itcompletely blocked proliferation of the wild-type DUB-1-expressing cells.

DISCUSSIONIn the present work, we describe a novel murine immediateearly gene that encodes a deubiquitinating enzyme, DUB-1.

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DUB-1C60S

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PROPIDIUM FLUORESCENCE

FIG. 5. Forced expression ofDUB-I results in growth arrest in theG, phase of the cell cycle. The indicated cell lines were grown for 48hr (5 x 105 cells/ml) with or without dexamethasone (0.1 j/M). Thecells were stained with propidium iodide and analyzed by flowcytometry. The percentage of cells in Gl, S, and G2/M were: DUB-I- dexamethasone (32%, 61%, 7%), + dexamethasone (82%, 14%,4%); DUB-I(C60S) - dexamethasone (35%, 57%, 8%), + dexameth-asone (35%, 57%, 8%). Data shown are representative of at least threeseparate dexamethasone induction experiments.

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Proc. Natl. Acad. Sci. USA 93 (1996) 3279

DUB-1 may specifically regulate proteolysis (or the ubiquiti-nation state) of a protein in an IL-3-specific signal transductionpathway. Identification of the specific substrates of DUB-1should help elucidate its mechanism of growth suppression.Constitutive expression of wild-type DUB-1 does not suppressthe growth of murine 3T3 fibroblasts (data not shown). Thissuggests that the growth suppression by DUB-1 might bespecific to hematopoietic cells, the only cell types in whichDUB-1 is normally expressed. This may reflect the existenceof a hematopoietic-specific substrate(s) of DUB-I, a hemato-poietic cell-restricted DUB-1 cofactor, or a higher threshold ofresistance to continuous DUB-1 expression in other cell types.Interestingly, we have isolated three additional genes whosepredicted products show high sequence similarity to DUB-1(approximately 80% amino acid identity) (Y.Z., unpublishedobservations). These genes are presumably DUB subfamilymembers, and we refer to them as DUB-2 through DUB-4.They may be induced by different growth factors and/or maydeubiquitinate different intracellular substrates.We hypothesize that, like other immediate early gene prod-

ucts, DUB-1 plays a role in integrating extracellular signals withcellular growth and cell cycle progression. Our data suggestthat turning off DUB-1 expression after a rapid burst ofIL-3-induced synthesis is crucial for hematopoietic cell prolif-eration. It is possible that after cytokine induction, cells onlyturn off DUB-1 under specific conditions, e.g., adequatenutrient availability, thereby providing a mechanism by whicha cell could halt cell cycle progression following exposure to amitogenic signal. Several other examples of mitogen-inducednegative regulators have recently been demonstrated. MKP-1(42) and PAC1 (43) are mitogen-induced threonine/tyrosinephosphatases that inactivate mitogen activating protein kinase.p21 is a mitogen-induced inhibitor of cyclin/cdk complexes(44). DUB-1 is the first enzyme of the ubiquitin system directlyimplicated in cytokine-regulated growth control.

We thank Peng Liang, Bernard Mathey-Prevot, David Pellman,Mark Ewen, Joan Ruderman, Chuck Stiles, and members of theD'Andrea laboratory for helpful discussions. We thank Barbara Keanefor preparation of the manuscript. This work was supported byNational Institutes of Health Grant RO1 DK 43889-01 (to A.D.D.).and Grant GM46904 (to M.H.). Y.Z. is a Fellow of the LeukemiaSociety of America. A.D.D. is a Lucille P. Markey Scholar and aScholar of the Leukemia Society of America, and this work was alsosupported in part by a grant from the Lucille P. Markey CharitableTrust.

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